Natural serine protease inhibitors play a central role in regulating serine protease activity. They are essential in maintaining homeostasis, and the improper or inadequate inactivation of serine proteases leads to a number of pathological states. Although much is known about the structure and function of several mammalian serine proteases and their corresponding inhibitors, little is known about the structure/function of protein inhibitors produced by parasitic organisms and their roles in the pathogenesis of parasitic infections. I hypothesize that, as a result of the ongoing biochemical warfare between hosts and their parasites, parasite inhibitors have undergone structural modifications which enable the parasite to inactivate potentially damaging host proteases, thus enhancing parasite survival. My lab has purified and partially characterized two elastase isoinhibitors from the infective stage of the ascarid nematode, Anisakis simplex. Amino acid sequence information reveals that these elastase isoinhibitors share identity with Ascaris chymotrypsin/elastase inhibitors and belong to a novel class of serine protease inhibitors. We have now cloned and are expressing these two Anisakis isoforms as well as the Ascaris chymotrypsin/elastase inhibitor. Anisakis and Ascaris, which are both members of the ascarid group of nematodes, each have their own distinct host range and life history patterns. The proposed study thus presents an opportunity to study the relationship of structural modifications to inhibitor function in the context of parasite adaptation to specific hosts. This study will also provide further insight into the mechanisms of protein-protein interaction and enrich our understanding of the structural determinant of this novel protein family. The Anisakis elastase inhibitors are excellent inhibitors of human leukocyte elastase (2-10 nm range), and may have potential as pharmacological agents in the treatment of inflammatory and vascular disorders. In this application, I propose to 1) determine the protease target specificities and biochemical properties of the Anisakis serine protease isoinhibitors (ASPIs) and compare them to the Ascaris inhibitor; 2) determine the 3-dimensional structure of the ASPIs to define the mechanism of inhibition and basis of inhibitory specificity and compare it to the x-ray crystal structure of the Ascaris inhibitor which has been solved by my collaborator, Dr. M.N.G. James, Univ., of Alberta; 3) characterize the Anisakis and Ascaris inhibitor gene families; 4) immunolocalize the protease inhibitors in larval Anisakis and Ascaris; and 5) expose these ascarid nematode larvae to their specific host's enzyme to further confirm the proposed biological function of these inhibitors.